The purpose of this proposal is to continue investigations exploring the biochemical and immunochemical differences between the life cycle stages (promastigote and amastigote) of the parasitic protozoan, Leishmania as these may provide important targets for chemotherapeutic or vaccine approaches for control. Work will be focused on amastigote-stage specific components. In the past period of support, considerable progress has been made in developing and characterizing lines of extracellular axenic amastigotes of Leishmania pifanoi, Leishmania braziliensis and Leishmania panamensis. In the current application, the axenic amastigotes will be used in conjunction with biochemical, immunological and newly developed genetic (molecular) approaches to further elucidate and explore the nature of and biological role for stage-specific molecules of this parasite system. As investigations to date of Leishmania have primarily focused on the promastigote stage of the parasite, this proposal represents a relatively unique and distinct area of investigation. Specifically, the aims are: 1.Molecular characterization of amastigote-specific molecules. Work will focus on the cloning, regulation and biochemical characterization of the P- 4, P-8, B-21-25 molecules. 2.Evaluation of amastigote specific antigens in eliciting protection against infection. Isolated amastigote molecules will be employed; studies will determine the nature of the T cell effector mechanisms involved; protective epitopes will be mapped. 3.The mechanisms involved in the generation of the megasome, a unique organelle of L. mexicana complex amastigotes will be investigated. Approaches will involve cell biology and biochemical studies of processing and targeting/sorting as well as newly developed genetic methods in Leishmania.